This invention relates to mutants of Neisseria useful for vaccine preparation. Specifically this invention relates to mutants of Neisseria containing mutations in a major outer membrane protein gene such that no immunologically functional polypeptides encoded by said gene are produced. More specifically, the invention relates to a mutant strain of Neisseria gonorrhoeae having a mutation of the PIII gene and to vaccines derived therefrom.
The genus Neisseria includes two species of gram-negative pyogenic cocci that are pathogenic to man: the meningococcus, Neisseria meningitidis, the causative agent of cerebrospinal meningitis, also referred to meningococcal meningitis and the gonococcus, Neisseria gonorrhoeae, the causative agent of the venereal disease gonorrhea.
Neisseria gonorrhoeae is an aerobic diplococcus that ferments glucose but not maltose, a characteristic useful in distinguishing the species from meningococci. Gonococci exhibit four colonial forms (T1-T4). Fresh isolates from clinical samples that retain their virulence grow as small colonies (T1 and T2). Repeated non-selective sub-culturing results in larger colonies (T3 and T4) which have been shown to be avirulent on inoculation in male volunteers.
Historically, the development of vaccines has been hampered by a number of technical problems inter alia, difficulty of cultivation, lack of a readily available and meaningful animal model, inability to classify the organisms by conventional serological techniques and the lack of protectiveness of whole bacterial cell vaccines when administered to humans.
Cell surface molecules are attractive candidates for vaccine compositions and have been extensively studied in bacteria in general and in Neisseria in particular.
Pili are proteinaceous, filamentous structures associated with the cell wall of infective strains of Neisseria. During infection the bacteria colonize the mucous membranes of the host; the attachment of the bacteria to the surface of the mucous membrane being mediated by the pili. Anti-pilin antibodies are thought to protect against infection by reacting with the pili and preventing the attachment of the bacterium to membrane target sites. Further the xe2x80x9ccoating actionxe2x80x9d of the antibody (i.e. opsonization) stimulates the removal of bacteria by phagocytic cells in the blood. U.S. Pat. Nos. 4,461,838 and 4,696,986 relate to crystalline and single rod structures derived from pili of Type 1 and Type 2Neisseria gonorrhoeae, methods for their preparation and the use of such material in vaccines.
One of the major shortcomings of pilin-based vaccines is the high antigenic variability associated with the pilin protein, thus antibodies raised against one strain will not necessarily react with any other strain. U.S. Pat. Nos., 4,584,195 and 4,622,223 and PCT application No. PCT/US85/00565 attempt to address this problem by employing as vaccine components, fragments of the pilin proteins which comprise conserved amino acid sequences, thus antisera raised thereto are characterized by a somewhat broader reactivity.
Protein I is found in the membranes of all gonococci and is usually present in the largest amount. The protein has been purified and when inserted into artificial bilayers acts as an anion-selective pore. There appear to be 14-20 serotypic PI variants, however only one PI is expressed by any one strain. Further certain PI variants have been associated specifically with pelvic inflammatory desease (P.I.D.) and other variants with disseminated gonococcal infection (D.G.I.). For these reasons PI has been used as a vaccine component,
U.S. Pat. Nos. 4,203,971 and 4,239,749 relate to methods of separating PI from outer membrane lipopolysaccharides which have been shown to have toxic properties and the use of the xe2x80x9cdetoxifiedxe2x80x9d product as a vaccine. European Patent Application No. 83301813.8 points out some of the shortcomings of the above patents, such as low yields and low solubility, thus making the production of effective vaccines difficult. The European application discloses an improved purification protocol which results in a protein I preparation having a solubility of from 5 to 10 mg/ml. As described, the vaccine also contains small amounts of protein II and protein III as well as what is characterized as xe2x80x9ctracexe2x80x9d amounts of LPS (3.5-4.0%). As discussed in more detail below, these contaminants although present in only small amounts can have a significant adverse effect on the effectiveness of PI vaccines.
When observed with light directly reflected from the substage mirror of a colony microscope, gonococcal colonies vary in their opacity: some appear transparent like water droplets, others are opaque like ground glass, and other colonies are intermediate in appearance. Gonococci change from the transparent to the opaque phenotype (or vice versa) with great frequency, estimated to be 10xe2x88x923 per cell division. The change to the opaque form is accompanied by the acquisition of one or more additional outer membrane proteins in the molecular weight range of 24,000-30,000. These proteins exhibit heat-modifiable behavior, namely, their apparent molecular weight on SDS-PAGE changes, depending on the degree to which they have been heated prior to electrophoresis. The optical property of opacity is due to the fact that the gonococci stick to each other within the colony and these zones of adhesion between outer membranes are mediated by the opaque proteins. This indicates that protein II binds to some constituent on the neighboring cell; however, this receptor-ligand relationship has not been defined. It has been noted that if all the colonial variants of a single strain are carefully examined as many as six different opaque proteins can be distinguished.
Although it is known that protein II is readily accessible to antibodies, very little information is available on the antibacterial effects that protein II antibodies might have. The limited serological data available currently indicate that these proteins are serologically specific, and the degree of cross-reactivity between them in the native state is not clearly delineated. The high degree of variability of this class of proteins has also discouraged interest in their use in a vaccine. Definitive estimates of the potential of protein II vaccines cannot be made until a much clearer picture emerges on their role (or quite likely multiple roles) in pathogenesis.
It has been shown that all gonococcal strains examined have an outer membrane protein that is not heat modifiable and that in the absence of a reducing agent migrates on SDS-PAGE with a molecular weight of 30,000, but with a molecular weight of 31,000 following reduction. This protein, named protein III, does not seem subject to variation, with all strains having an identical protein as judged by peptide analysis. A hybridoma has been cloned that produces and antibody reactive with protein III as it exists in the membrane, indicating that at least one antigenic determinant is exposed to the surface (Swanson, J. et al., Infect. Immunity 38: 668-672 (1982)). In vivo cross-linking studies have shown that protein III is closely associated with the trimeric complex of protein (McDade R. L., et al., J. Bacteriol. 141: 1183-1191 (1980)).
The ubiquity and constancy of PIII initially made it an attractive candidate for vaccine, however, recent studies raise serious questions about the utility of PIII as a vaccine. For example, Rice, P. A. et al., (J. Exp. Med. 164: 1735-1748 (1986)) disclose that IgG antibodies directed against PIII act to block the killing of serum-resistant Neisseria by immune sera. As a result of eliciting these blocking antibodies, PIII quite paradoxically helps to protect the gonococcus from attack by antibodies to other surface antigens. Because it is technically very difficult to remove PIII from gonococcal membrane antigen preparations, vaccinations with PI results in, if anything, a diminished protective effect. (Arminson, P., Abst. Ann. Meeting American Society for Microbiology, 118 (1987)).
U.S. Pat. No. 4,220,638 and its cognate European Application No. 78400245.3 disclose the isolation and use of a macromolecular aggregate as a vaccine. The aggregate is characterized as being derived from the cell surface of Neisseria, having a molecular weight of 9.2xc3x97106 and having 5 major components which account for 80% by weight of the complex. One of the components has a molecular weight (xcx9c27,000) that is in the range of that reported for PIII (xcx9c30,000).
U.S. Pat. No. 4,330,623 relates to a method of solubilizing gonococcal antigens such as the macromolecular complex described above by treatment with trypsin.
The lipopolysaccharide (LPS) of the gonococcus (like that of the meningococcus) consists of only short oligosaccharies attached to a lipid A moiety. The sugars hat have been detected include KDO, glucose, galactose, heptose, glucosamine, and galactosamine. Studies on the antigenic properties of LPS indicate that there are determinants common to all gonococci that can be demonstrated using antiserum raised in hens (Wallace, R. et al., Can. J. Microbiol. 24: 124-128 (1978)) or monoclonal antibodies. However, there are also LPS serological determinants that allow classification of different gonococcal strains into six types (Apicella, M. et al., Infect. Immun. 26: 870-74 (1979)).
U.S. Pat. No. 4,681,761 relates to methods of purifying the major iron-regulated protein (MIRP) of N. gonorrhoeae and its use as a vaccine.
U.S. Pat. No. 4,351,761 discloses the purification an properties of the so-called xe2x80x9cL-antigenxe2x80x9d from Neisseria gonorrhoeae and its use in a diagnostic test to identify the presence of antibodies in human sera to N. gonorrhoeae. 
U.S. Pat. No. 4,707,543 relates to a process for preparing a detoxified polysaccharidexe2x80x94outer membrane protein complex and its use as a vaccine.
European Patent Application No. 85201657.5 relates to a vaccine composition in which the antigenic activity of the protein is enhanced by the formulation of an absorbed antigen-detergent complex.
In an alternative approach, immunological reagents reactive with the pilus receptors on epithelial cells have been investigated as exemplified by European Patent Applications Nos. 83850077.5 and 86401916.1.
McShan, W. M. et al., (Infection and Immunity 55 (12): 3017-3022 (1987)) disclose the cloning and expression of genetic information derived from a serum resistant strain of gonococcus that confers serum resistance upon a serum sensitive strain. The DNA encodes a protein that can mediate the attachment of a blocking antibody to the cell surface. This blocking antibody interferes with the complement-mediated bactericidial antibody activity. Although similar in size, the protein, as the reference clearly shows, is antigenically unrelated to PIII.
Koomey, J. M. et al., (Proc. Nat""l. Acad. Sci (USA). 79(12): 7881-7885 (1982)), disclose the cloning and expression of a gonococcal gene (IgAl protease) in E. coli. The article also discloses deleting a portion of the cloned gene and inserting a marker gene therein. The resulting DNA is incapable of expressing the cloned gene product. This DNA is then used to transform normal gonococci to yield mutant strains in which the defective gene has been integrated into the bacterial chromosome by a double-crossover event.
Finally, a large number of patents exist which relate to the diagnosis of Neisseria. See, for example, U.S. Pat. Nos. 4,208,480, 4,241,045, 4,446,230, 4,497,900 and 4,659,658.
The invention relates to novel mutants of Neisseria gonorrhoeae and the use of such strains in the preparation of an improved vaccine for the prevention of gonococcal infections. More specifically the invention relates to strains of N. gonorrhoeae in which the gene for Protein III (PIII) has been inactivated. PIII is a highly conserved, antigenically stable gonococcal outer membrane protein. Although PIII appears to be an ideal candidate as an active ingredient in a vaccine, it has been demonstrated that the antibodies elicited in response to PIII actually inhibit the bactericidal activity of immune serum against gonococci. These so-called blocking antibodies seriously limit the effectiveness of prior art vaccines derived from outer membrane components because an antigenically significant amount of PIII co-purifies with the desired antigen even in the case of relatively pure antigen preparations. The development of a strain incapable of producing immunologically functional PIII provides a useful source of other outer membrane antigens without the problem of PIII contamination.
The subject invention may be viewed as having several embodiments. The invention relates to a biologically pure culture of a mutant strain of the genus Neisseria incapable of producing a protein that elicits blocking antibodies or immunologically reacts with said antibodies. In a further embodiment the invention relates to a vaccine comprising an immunologically effective amount of an antigen derived from a strain of Neisseria incapable of producing immunologically functional PIII-like protein admixed with a physiologically acceptable excipient. In a yet another embodiment the invention relates to an antigen substantially free of PIII-like protein obtained from a strain of Neisseria incapable of producing PIII-like protein. In yet another embodiment the invention relates to an improvement in a method of preparing a Neisseria antigen suitable for vaccine preparation comprising the steps of culturing a strain of Neisseria and recovering a cell surface antigen therefrom, the improvement which comprises culturing a strain of Neisseria substantially incapable of producing a PIII-like protein. In yet another embodiment the invention relates to an improvement in a method for the immunization of a mammal against Neisseria infection by employing a vaccine comprising an antigen derived from the cell surface of Neisseria; the improvement which comprises utilizing as a source of the antigen a strain of Neisseria substantially incapable of producing a PIII-like protein. In a final embodiment the invention provides a proteosome preparation in which the protein is derived from a strain of Neisseria incapable of producing immunologically functional PIII-like protein.